The mechanism of EphB4/ephrinB2 in the resistance of chronic myelogenous leukemia to imatinib keeps unknown. The imatinib resistant chronic myelogenous leukemia cell line-K562-R, was established. EphB4 receptor expression was detected in patients and resistant cells. Cell migration and drug sensitivity were tested in the EphB4 knockdown cells and mouse models.
Int J Med Sci 2016, Vol 13 Ivyspring International Publisher 365 International Journal of Medical Sciences 2016; 13(5): 365-373 doi: 10.7150/ijms.14989 Research Paper EphB4/ephrinB2 Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Involved in Cytoskeletal Proteins Lin Li1,*, Na Xu1,*, Jin-fang Zhang2, Lu-lu Xu1, Xuan Zhou1, Bin-tao Huang3, Yu-ling Li1, Xiao-li Liu1, Department of Hematology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou 510515, Guangdong, China Department of Paediatric Hematology and Oncology, Clinical Center of Tumor Therapy, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 510000, China Department of Hematology, The Affiliated Hospital of Inner Mongolia Medical University, Tongdao Avenue North, Hohhot 010059, China * Lin Li and Na Xu contributed equally to this work and should be considered as co-first authors Corresponding author: Xiao-li Liu, Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China Phone: +86 (020) 6164-1616; Fax: +86 (020) 6164-1616; E-mail: lxl2405@126.com © Ivyspring International Publisher Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited See http://ivyspring.com/terms for terms and conditions Received: 2016.01.16; Accepted: 2016.04.12; Published: 2016.04.28 Abstract Introduction: The mechanism of EphB4/ephrinB2 in the resistance of chronic myelogenous leukemia to imatinib keeps unknown Methods: The imatinib resistant chronic myelogenous leukemia cell line-K562-R, was established EphB4 receptor expression was detected in patients and resistant cells Cell migration and drug sensitivity were tested in the EphB4 knockdown cells and mouse models Results: The EphB4 receptor was over-expressed in blast crisis patients compared to chronic phase patients The level of EphB4 receptor expression was associated with a complete cytogenetic response within 12 months Enhanced expression of the EphB4 receptor was detected in the K562-R cells EphB4 knockdown inhibited cell migration ability and restored sensitivity to imatinib in vitro and in vivo Restored sensitivity to imatinib was observed in K562-R cells, along with increased levels of phospho-EphB4 and decreased phosphorylation levels of RhoA, Rac1, and Cdc42 Conclusion: Our study illustrates that aberrant activation of EphB4/ephrinB2 may mediate chronic myeloid leukemia resistance involved in cytoskeletal proteins Key words: Chronic myelogenous leukemia; imatinib; EphB4/ephrin B2; shRNA Introduction Imatinib (IM) is used as the standard chemotherapy drug for many chronic myelogenous leukemia (CML) patients and can significantly prolong a patient’s survival However, IM drug resistance occurs in 20-30% of patients and results in a progressive outcome Although second generation kinase inhibitors can overcome this BCR-ABL-dependent resistance, their effects are limited [1] TKI-resistant CML is more complex than the original spontaneous IM resistance BCR-ABL-dependence also contributes to resistance [2] Signal transduction through the erythropoietin producing hepatoma (Eph) amplified sequence receptors binding to their cell-surface ephrin ligands and have been implicated in hematopoiesis and the growth of various cancer cells [3].The binding of an ephrin ligand to its respective Eph receptor activates a bi-directional signal (as well as cell adhesion), thereby affecting cell proliferation and cell-fate determination in a number of key biological processes, including angiogenesis and hematopoiesis [4-6] The Eph receptors (which are phosphorylated on two conserved tyrosines) enable efficient kinase activation http://www.medsci.org Int J Med Sci 2016, Vol 13 in the juxta-membrane domain, which modulates many adaptors and effectors including non-receptor tyrosine kinases of the Src and Abl families (particularly Rho and Ras family GTPases) This activation may also regulate the molecular expression of other receptor tyrosine kinase (RTK)family members [5, 7, 8] These crucial regulators assist most RTK families in controlling cell growth and migration, which ph receptors use to inhibit cell proliferation and survival Some studies have indicated that ephrinB2-Fc activated EphB4 receptors could decrease cell viability and suppress cell invasion in breast and colon cancer [9-11] Previous reports have also found that increased EphB4 expression levels are related to poor prognoses in breast cancer [12] Recent findings have shown that EphB4/ephrinB2 signaling may be involved in the pathology of leukemia [3], and over expression of EphB4 has been associated with drug resistance in Ph+ acute lymphocytic leukemia (ALL) [13] It also has been shown that EphB4 promotes cell adhesion-mediated drug resistance by interacting with the extracellular matrix and modulating Rho family members Our previous study has established the EphB4 knockdown and IM resistant CML cell line (K562-R-EphB4-sh) and found that homoharringtonine overcomes imatinib resistance by blocking the EphB4/RhoA pathway in K562-R [14] To determine the role and mechanism of EphB4/ephrinB2 in IM resistance to CML, we investigated the role of EphB4 in IM resistant CML, and we further explored the related molecules and the down-stream signaling pathway of EphB4/ephrinB2 Materials and Methods Study Population Clinical samples were taken from the bone marrow of chronic myelogenous leukemia chronic phase (CML-CP) patients (n=22), as well as chronic myelogenous leukemia blast crisis (CML-BC) patients (n=6) to produce bone marrow (BM) cells in the first diagnosis All of the patients had received IM therapy, and CML-BC patients were resistant to imatinib The clinical diagnosis of CML was evaluated by the National Comprehensive Cancer Network All patients agreed to provide their written informed consent for the use of their samples The protocol was approved by the Ethics Committee of the Nanfang Hospital, Southern Medical University Cell Culture K562 wild type cells (American Type Culture Collection, Manassas, VA, USA) were cultured in a RPMI-1640 medium supplemented with 10% Fetal 366 Bovine Serum (FBS)(Sigma-Aldrich, St Louis, MO, USA) at 37˚C, 5% CO2 K562 wild type cells were incubated with IM (initial at 0.05 µM) to establish IM resistance using the step-wise method described previously [14] IM (Sigma, USA) concentration was increased by 0.05 µM every ten or twenty days until reaching 2.8 µM The surviving cells were then cultured in RPMI-1640/10% FBS at 37˚C, 5% CO2 in the absence of IM for approximately three days Cells were re-examined for sensitivity to IM and resistant K562 cells were labeled as K562-R K562-R-EphB4-sh represented the established IM resistant cells with EphB4 knockdown by shRNA from our previous study [14] Real Time PCR Total RNA of patient samples and cell lines was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and stored at -80˚C All RT-PCR primers were designed with Primer 5.0 and listed below: EphB4 forward primer: 5’-ACCTCCATTCCT GCGGCTAA-3’; Reverse primer: 5’-AGACGAGGTT GCTGTTGACT-3’ GAPDH forward primer: 5’-GAGGGGTGAT GTGGGGAGTA-3’; Reverse primer: 5’-GAGCTTCC CGTTCAGCTCAG-3’ PCR conditions were: initial 95˚C, 15 min; denaturation 94˚C, 15 s; annealing 60˚C 30 s and extension 72˚C, 30 s The number of cycles was 35-45 Western blot analysis Proteins were firstly extracted using the RIPA lysis buffer with adding halt protease and the phosphatase inhibitor (Thermo Fisher Scientific, Boston, MA, USA) The concentrations of proteins were measured with the bicinchoninic acid (BCA) method Proteins were then separated by SDS-PAGE and transferred onto nitrocellulose membranes following hours blocking with 5% bovine serum albumin (BSA) The membrane was incubated with primary antibody at 4˚C overnight and then continually incubated with HRP-conjugated goat anti-rabbit (1:2000) or goat anti-mouse (1:5000) (Cell signaling, Berverly, MA, USA) immunoglobulin diluted in 5% BSA for l hour Blots were washed again, followed by an enhanced chemiluminescence assay (Cell signaling) to visualize the secondary antibody Apoptosis assessment and cell cycle assay K562, K562-R-EphB4-sh, and K562-R cells were collected, washed twice, and incubated with PE Annexin V in a buffer containing 7-AAD for 15 at room temperature (BD Pharminigen, San Diego, CA, USA.) Cells were then examined by flow cytometry http://www.medsci.org Int J Med Sci 2016, Vol 13 Cells (5×105 cells/ml) were seeded in 24-well plates and cultured in l ml media for 24 hours The exponentially growing cells were then incubated with IM (0.5 µM) Cells were pelleted and then fixed for h at 4˚C by adding 500 µL of fixation solution The fixed cells were pelleted and analyzed by flow cytometry (FACS)(BD Pharminigen) Cell migration assay Cell migration was detected with commercial transwell assay (Corning, Tewksbury, MA, USA) Briefly, 10% (v/v) fetal calf serum 1640-mediumwas added to the lower chamber, and 3×104cells were suspended in serum-free media and added to the upper chamber following h incubation supplied with 5% CO2 at room temperature Then, the upper chamber was detached from the assay, and the cells were collected from the bottom chamber Total number of cells was counted at three randomly selected fields Cell viability assay Cell viability of K562, K562-R-phB4-sh, and K562-R cells were measured using a cell counting kit-8 (CCK-8) (Sigma) 5×104 cells/well were seeding into 96-well micro-plate containing in 100 µl media and followed a series of concentrations of IM (0-8 µM) treatment After 24 hours of culture, 10 µl CCK-8 solution was added into each well and incubated for hours at room temperature Cell viability was detected using a microplate reader (Bio-rad 680, Hercules, CA, USA) at 450nm optical density (OD) and quantified as [(OD treated -OD blank)/(ODcontrol-OD blank]× 100% The IC50 value of each cell line was calculated from the cell inhibition rate Xenograft models K562 parental cells, K562-R-EphB4-sh, and K562-R cells (1x107/ml) were subcutaneously injected into BALB/C nude mice, (female nude mice 4-5 weeks of age; SLRC laboratory animal center, China) to build xenograft models On day 30 after implantation, tumors reached an average size of over 1000 mm As determined by histopathology, green fluorescent protein (GFP) positive cells in the xenograft tumor were found in the K562-R-EphB4-sh cell group Then, all nude mice received an oral standard-dose of IM (91 µg/g daily for 30 days) treatment All animal experiments were conducted according to the policy and procedures provided by the Committee for Animal Research and Ethics and were approved by the Ethics Committee of Nanfang Hospital, Southern Medical University Enzyme-linked immunosorbent assay (ELISA) The phosphorylation level of EphB4 in each cell 367 line was detected by phosphorylation ELISA (Abcam, Cambridge, UK) The anti-human EphB4 antibody specifically recognizes and binds human EphB4 proteins (including the phosphorylated and non-phosphorylated proteins) and was immobilized on 96-well micro titer plates as the capture antibody Cell lysate was then added to the plates Plates were washed to remove unbound proteins, and HRP-conjugated anti-tyrosine phosphorylated antibodies specific for phosphorylated proteins were added After the plates were washed again, chromogenic substrate was added and the absorbance of the substrate was measured at 450 nm Statistical analysis Data were presented with the mean ± standard deviation (SD) from three independent experiments SPSS13.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis Paired t test or LSD method were used for the comparison between groups and within groups Pearson correlation coefficients were performed to assess the correlation between EphB4 mRNA and BCR-ABL transcript levels P < 0.05 was considered to be statistically significant Results Association of EphB4 expression with clinical stage and IM drug response in CML patients The expression levels of EphB4 from CML-CP and CML-BC patients are shown in Fig 1A Our results also show that EphB4 mRNA levels in CML patients significantly increased according to clinical stages (P